摘要
基于PTEN诱导的激酶1(PINK1)/帕金森病蛋白(Parkin)通路研究加味独活寄生合剂对膝骨关节炎(KOA)兔软骨线粒体自噬的影响,探讨其改善软骨病变的可能机制。通过在兔右侧后肢固定高分子树脂石膏绷带建立KOA模型,周期6周。造模成功后,随机将造模组分为模型组,塞来昔布组,加味独活寄生合剂低、高剂量组,每组8只。塞来昔布组给予塞来昔布灌胃,单次剂量0.0093 g·kg-1;加味独活寄生合剂低、高剂量组分别给予加味独活寄生合剂单次生药量6.8、27.2 mL·kg-1,即4.5152、18.0608 g·kg-1灌胃,每日1次,连续灌胃6周后对各组兔进行行为学检测,取材后肉眼观察各组兔膝关节软骨大体形态改变,苏木素-伊红(HE)染色检测软骨组织病理学变化,并通过Lequesne MG评分、Pelletier评分及Mankin评分进行量化评估;透射电镜检测软骨线粒体超微形态变化,流式细胞仪检测软骨细胞线粒体膜电位(Δψm)和活性氧(ROS)平均荧光强度并计算低Δψm细胞百分比,蛋白免疫印迹法检测软骨组织线粒体自噬相关蛋白PINK1、Parkin、选择性自噬接头蛋白(P62)、微管蛋白1轻链3(LC3)Ⅱ/LC3Ⅰ、线粒体外膜转位酶20(TOM20)、Ⅱ型胶原蛋白α1(COL2A1)、软骨蛋白聚糖抗体(ACAN)、基质金属蛋白酶(MMP)-9、MMP-13表达水平,免疫组化检测软骨组织PINK1、Parkin、LC3B表达。结果显示,与空白组相比,模型组兔膝关节出现明显肿胀破坏,组织纤维化,软骨细胞分布松散,潮线模糊、不完整,Lequesne MG评分、Pelletier评分及Mankin评分显著升高,自噬小体减少,线粒体形态异常,软骨细胞低Δψm细胞百分比、ROS平均荧光强度以及软骨组织P62、TOM20、MMP-9、MMP-13蛋白表达显著升高,软骨组织PINK1、Parkin、LC3Ⅱ/LC3Ⅰ、COL2A1、ACAN蛋白表达显著降低。与模型组相比,塞来昔布组、加味独活寄生合剂各剂量组兔膝关节软骨表面破坏均有所改善,软骨组织潮线相对完整,软骨细胞数量相对密集,Lequesne MG评分、Pelletier评分及Mankin评分均显著降低,自噬小体增多,线粒体肿胀、损伤情况出现好转,软骨细胞低Δψm细胞百分比、ROS平均荧光强度以及软骨组织P62、TOM20、MMP-9、MMP-13蛋白表达显著降低,软骨组织PINK1、Parkin、LC3Ⅱ/LC3Ⅰ、COL2A1、ACAN蛋白表达显著升高。综上,加味独活寄生合剂可有效激活PINK1/Parkin通路从而促进线粒体自噬,改善KOA兔关节软骨损伤,从而延缓KOA的进展。
This study aims to investigate the effects of Jiawei Duhuo Jisheng Mixture on mitochondrial autophagy in the cartilage of rabbits with knee osteoarthritis(KOA)based on the PTEN-induced kinase 1(PINK1)/Parkinson protein(Parkin)pathway and explore its potential mechanism in improving cartilage lesions.A KOA model was established by fixing a high-molecular resin plaster bandage on the right hind limb of the rabbits for six weeks.After successful modeling,the modeling group was randomly divided into a model group,a celecoxib group,and low-and high-dose groups of Jiawei Duhuo Jisheng Mixture,with eight rabbits in each group.The celecoxib group was administered celecoxib by gavage at a single dose of 0.0093 g·kg-1.The low-and high-dose groups of Jiawei Duhuo Jisheng Mixture were given Jiawei Duhuo Jisheng Mixture at single doses of 6.8 mL·kg-1(4.5152 g·kg-1)and 27.2 mL·kg-1(18.0608 g·kg-1),respectively.Administered once daily for six weeks,the rabbits in each group then underwent behavioral testing.After sample collection,the gross morphological changes of the knee articular cartilage were observed with the naked eye.Hematoxylin-eosin(HE)staining was used to detect pathological changes in cartilage tissue,which were quantitatively evaluated by using the Lequesne MG score,Pelletier score,and Mankin score.Transmission electron microscopy was used to observe the ultrastructural changes of chondrocyte mitochondria.Flow cytometry was used to detect the mitochondrial membrane potential(Δψm)and the average fluorescence intensity of reactive oxygen species(ROS)in chondrocytes and calculate the percentage of cells with lowΔψm.Western blot was used to detect the expression level of mitochondrial autophagy-related proteins in cartilage tissue,including PINK1,Parkin,selective autophagy adapter protein 62(P62),light chain 3(LC3)Ⅱ/LCⅠ,mitochondrial outer membrane translocase 20(TOM20),collagen typeⅡalpha 1(COL2A1),aggrecan(ACAN),matrix metalloproteinase(MMP)-9,and MMP-13.Immunohistochemistry(IHC)was used to detect the expression of PINK1,Parkin,and LC3B in cartilage tissue.The results showed that,compared with the blank group,the model group exhibited marked knee joint swelling and damage,tissue fibrosis,sparse chondrocyte distribution,and indistinct and incomplete tide marks.The Lequesne MG,Pelletier,and Mankin scores increased significantly.Autophagosomes were reduced,and mitochondria were morphologically abnormal.The percentage of chondrocytes with lowΔψm,ROS average fluorescence intensity,and the expression of P62,TOM20,MMP-9,and MMP-13 proteins in cartilage tissue rose significantly,while the expression of PINK1,Parkin,LC3Ⅱ/LCⅠ,COL2A1,and ACAN proteins in cartilage tissue decreased significantly.Compared with the model group,the celecoxib and both Jiawei Duhuo Jisheng Mixture groups showed improved knee articular cartilage surface,relatively intact tide marks,and denser chondrocytes.Their Lequesne MG,Pelletier,and Mankin scores dropped signicantly.Autophagosomes increased,and mitochondrial swelling and damage eased.The percentage of chondrocytes with lowΔψm,ROS average fluorescence intensity,and the expression of P62,TOM20,MMP-9,and MMP-13 proteins in cartilage tissue decreased significantly,while the expression of PINK1,Parkin,LC3Ⅱ/LCⅠ,COL2A1,and ACAN proteins in cartilage tissue increased significantly.In conclusion,Jiawei Duhuo Jisheng Mixture can effectively activate the PINK1/Parkin pathway to promote mitochondrial autophagy and alleviate articular cartilage damage in rabbits with KOA,thus slowing the progression of KOA.
作者
叶子丰
院一蔚
文志
谭旭仪
欧梁
许晓彤
邝高艳
卢敏
YE Zi-feng;YUAN Yi-wei;WEN Zhi;TAN Xu-yi;OU Liang;XU Xiao-tong;KUANG Gao-yan;LU Min(Hunan University of Chinese Medicine,Changsha 410208,China;the First Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine,Changsha 410006,China)
出处
《中国中药杂志》
北大核心
2025年第22期6391-6399,共9页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(82174414,82274543,82405447)
湖南省科技创新计划项目(2022RC1225)
湖南省中医药科研课题(A2023012)。
